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Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genetically encoded Förster resonance energy transfer (FRET)-based tension sensors measure piconewton-scale forces across individual molecules in living cells or whole organisms. These biosensors show comparably high FRET efficiencies in the absence of tension, but FRET quickly decreases when forces are applied. In this article, we describe how such biosensors can be generated for a specific protein of interest, and we discuss controls to confirm that the observed differences in FRET efficiency reflect changes in molecular tension. These FRET efficiency changes can be related to mechanical forces as the FRET-force relationship of the employed tension sensor modules are calibrated. We provide information on construct generation, expression in cells, and image acquisition using live-cell fluorescence lifetime imaging microscopy (FLIM). Moreover, we describe how to analyze, statistically evaluate, and interpret the resulting data sets. Together, these protocols should enable the reader to plan, execute, and interpret FRET-based tension sensor experiments. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

The ability of cells to sense and respond to mechanical forces is crucial for a wide range of developmental and pathophysiological processes. The molecular mechanisms underlying cellular mechanotransduction, however, are largely unknown because suitable techniques to measure mechanical forces across individual molecules in cells have been missing. In this article, we highlight advances in the development of molecular force sensing techniques and discuss our recently expanded set of FRET-based tension sensors that allows the analysis of mechanical forces with piconewton sensitivity in cells. In addition, we provide a theoretical framework for the design of additional tension sensor modules with adjusted force sensitivity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The inability to measure mechanical forces within cells has been limiting our understanding of how mechanical information is processed on the molecular level. In this chapter, we describe a method that allows the analysis of force propagation across distinct proteins within living cells using Förster resonance energy transfer (FRET)-based biosensors.

Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle–tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo. [ABSTRACT FROM AUTHOR]

The ability of cells to sense and respond to mechanical forces is central to a wide range of biological processes and plays an important role in numerous pathologies. The molecular mechanisms underlying cellular mechanotransduction, however, have remained largely elusive because suitable methods to investigate subcellular force propagation were missing. Here, we review recent advances in the development of biosensors that allow molecular force measurements. We describe the underlying principle of currently available techniques and propose a strategy to systematically evaluate new Förster resonance energy transfer (FRET)-based biosensors. [ABSTRACT FROM AUTHOR]

The development of calibrated Förster resonance energy transfer (FRET)-based tension sensors has allowed the first analyses of mechanical processes with piconewton (pN) sensitivity in cells. Here, we introduce the working principle of this emerging microscopy method and discuss how it has been utilized to obtain quantitative insights into the mechanisms of intracellular force transduction in cell–matrix adhesions, cell–cell junctions, and at the cell cortex. These examples demonstrate that genetically encoded tension sensors are powerful tools to unravel force transduction mechanisms, but also indicate current limitations. We propose that further technical improvements are needed to develop a truly molecular understanding of mechanobiological processes in cells and tissues. [ABSTRACT FROM AUTHOR]